2,3-Olefinic epothilone derivatives

ABSTRACT

The present invention relates to 2,3-position modified epothilone derivatives, methods of preparation of the derivatives, and intermediates therefor. The compounds of the invention as 16-membered macrolides having the general structure,  
                 
 
     which have microtubule-stabilizing effects and cytotoxic activity against rapidly proliferating cells, such as, tunior cells or other hyperproliferative cellular disease.

FIELD OF THE INVENTION

[0001] The present invention relates to epothilone derivatives, methodsfor the preparation of the derivatives and intermediates therefor.

BACKGROUND OF THE INVENTION

[0002] Epothilones are macrolide compounds which find utility in thepharmaceutical field. For example, Epothilones A and B having thestructures:

[0003] Epothilone A R=H

[0004] Epothilone B R=Me

[0005] have been found to exert microtubule-stabilizing effects similarto TAXOL and hence cytotoxic activity against rapidly proliferatingcells, such as, tumor cells or other hyperproliferative cellulardisease, see Angew. Chem. Int. Ed. Engl., 1996, 35, No. 13/14.

SUMMARY OF THE INVENTION

[0006] The present invention relates to compounds of the formula

[0007] Q is selected from the group consisting of

[0008] G is selected from the group consisting of alkyl, substitutedalkyl, aryl, substituted aryl, heterocyclo,

[0009] W is O or N R₁₂;

[0010] X is O, S, or H, H;

[0011] Y is selected from the group consisting of O; H, OR₁₃; OR₁₄,OR₁₄; NOR₁₅; H, NOR₁₆; H, NR₁₇R₁₈; H, H; or CHR₁₉; OR₁₄ OR₁₄ can be acyclic ketal;

[0012] B is selected from the group consisting of H, OR₂₀, or OCOR₂₁,and NR₂₂R₂₃;

[0013] D is selected from the group consisting of NR₂₄R₂₅ or saturatedheterocycle (such as piperidinyl, pyrrolidinyl, and the like);

[0014] R₁, R₂, R₃, and R₄ are selected from H or lower alkyl;

[0015] R₁₅, R₁₆, R₁₇, R₁₈, and R₁₉ are selected from the group H, alkyl,substituted alkyl, or aryl;

[0016] R₆, R₇, R₁₃, R1₄, R₂₀, and R₂₁ are selected from the group H,alkyl, or substituted alkyl;

[0017] R₅, R₈, R₉, R₂₂, R₂₄, R₂₆, and R₂₇ are selected from the groupconsisting of H, alkyl, substituted alkyl, aryl, heteroaryl, cycloalkyl,or heterocyclo;

[0018] R₁₂, R₂₃, and R₂₅ are selected from the group consisting of H,alkyl, substituted alkyl, aryl, heteroaryl, cycloalkyl, heterocyclo,R₂₆C=O, R₂₇SO₂, hydroxy, O-alkyl or O-substituted alkyl;

[0019] and any salts, solvates or hydrates thereof

[0020] Proviso

[0021] The present invention does not include compounds of formula Iwherein

[0022] W and X are both O; and

[0023] R₁, R₂, R₃, R₄ are methyl; and

[0024] R₅ is H or methyl; and

[0025] G is 1-methyl-2-(2-methyl-4-thiazolyl)ethenyl; and

[0026] Q is

DETAILED DESCRIPTION OF THE INVENTION

[0027] Listed below are definitions of various terms used to describethis invention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

[0028] The term “alkyl” refers to straight or branched chainunsubstituted hydrocarbon groups of 1 to 20 carbon atoms, preferably 1to 7 carbon atoms. The expression “lower alkyl” refers to unsubstitutedalkyl groups of 1 to 4 carbon atoms.

[0029] The term “substituted alkyl” refers to an alkyl group substitutedby, for example, one to four substituents, such as, halo,trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyoxy,heterocylooxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino,arylamino, aralkylamino, cycloalkylamino, heterocycloamino,disubstituted amines in which the 2 amino substituents are selected fromalkyl, aryl or aralkyl, alkanoylamino, aroylamino, aralkanoylamino,substituted alkanoylamino, substituted arylamino, substitutedaralkanoylamino, thiol, alkylthio, arylthio, aralkylthio,cycloalkylthio, heterocyclothio, alkylthiono, arylthiono, aralkylthiono,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, sulfonamido (e.g. SO₂NH₂),substituted sulfonamido, nitro, cyano, carboxy, carbamyl (e.g. CONH₂),substituted carbamyl (e.g. CONH alkyl, CONH aryl, CONH aralkyl or caseswhere there are two substituents on the nitrogen selected from alkyl,aryl or aralkyl), alkoxycarbonyl, aryl, substituted aryl, guanidino andheterocyclos, such as, indolyl, imidazolyl, furyl, thienyl, thiazolyl,pyrrolidyl, pyridyl, pyrimidyl and the like. Where noted above where thesubstituent is further substituted it will be with halogen, alkyl,alkoxy, aryl or aralkyl.

[0030] The term “halogen” or “halo” refers to fluorine, chlorine,bromine and iodine.

[0031] The term “aryl” refers to monocyclic or bicyclic aromatichydrocarbon groups having 6 to 12 carbon atoms in the ring portion, suchas phenyl, naphthyl, biphenyl and diphenyl groups, each of which may besubstituted.

[0032] The term “aralkyl” refers to an aryl group bonded directlythrough an alkyl group, such as benzyl.

[0033] The term “substituted aryl” refers to an aryl group substitutedby, for example, one to four substituents such as alkyl; substitutedalkyl, phenyl, substituted phenyl, heterocyclo, halo, trifluoromethoxy,trifluoromethyl, hydroxy, alkoxy, cycloalkyloxy, heterocyclooxy,alkanoyl, alkanoyloxy, amino, alkylamino, aralkylamino, cycloalkylamino,heterocycloamino, dialkylamino, alkanoylamino, thiol, alkylthio,cycloalkylthio, heterocyclothio, ureido, nitro, cyano, carboxy,carboxyalkyl, carbamyl, alkoxycarbonyl, alkylthiono, arylthiono,alkysulfonyl, sulfonamido, aryloxy and the like. The substituent may befurther substituted by halo, hydroxy, alkyl, alkoxy, aryl, substitutedaryl, substituted alkyl or aralkyl.

[0034] The term “cycloalkyl” refers to optionally substituted, saturatedcyclic hydrocarbon ring systems, preferably containing 1 to 3 rings and3 to 7 carbons per ring which may be further fused with an unsaturatedC₃-C₇ carbocyclic ring. Exemplary groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,cyclodecyl, cyclododecyl, and adamantyl. Exemplary substituents includeone or more alkyl groups as described above, or one or more groupsdescribed above as alkyl substituents.

[0035] The terms “heterocycle”, “heterocyclic” and “heterocyclo” referto an optionally substituted, fully saturated or unsaturated, aromaticor nonaromatic cyclic group, for example, which is a 4 to 7 memberedmonocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclicring system, which has at least one heteroatom in at least one carbonatom-containing ring. Each ring of the heterocyclic group containing aheteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen atoms,oxygen atoms and sulfur atoms, where the nitrogen and sulfur heteroatomsmay also optionally be oxidized and the nitrogen heteroatoms may alsooptionally be quaternized. The heterocyclic group may be attached at anyheteroatom or carbon atom.

[0036] Exemplary monocyclic heterocyclic groups include pyrrolidinyl,pyrrolyl, indolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl,imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl,isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl,piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,2-oxopyrrolidinyl, 2-oxazepinyl, azepinyl, 4-piperidonyl, pyridyl,N-oxo-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl,tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl,thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone,1,3-dioxolane and tetrahydro-1, 1-dioxothienyl, dioxanyl,isothiazolidinyl, thietanyl, thiiranyl, triazinyl, and triazolyl, andthe like.

[0037] Exemplary bicyclic heterocyclic groups include benzothiazolyl,benzoxazolyl, benzothienyl, quinuclidinyl, quinolinyl,quinolinyl-N-oxide, tetrahydroisoquinolinyl, isoquinolinyl,benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl,coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl,furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,1-b]pyridinyl] orfuro[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as3,4-dihydro-4-oxo-quinazolinyl), benzisothiazolyl, benzisoxazolyl,benzodiazinyl, benzofurazanyl, benzothiopyranyl, benzotriazolyl,benzpyrazolyl, dihydrobenzofuryl, dihydrobenzothienyl,dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone,dihydrobenzopyranyl, indolinyl, isochromanyl, isoindolinyl,naphthyridinyl, phthalazinyl, piperonyl, purinyl, pyridopyridyl,quinazolinyl, tetrahydroquinolinyl, thienofuryl, thienopyridyl,thienothienyl, and the like.

[0038] Exemplary substituents include one or more alkyl groups asdescribed above or one or more groups described above as alkylsubstituents. Also included are smaller heterocyclos, such as, epoxidesand aziridines.

[0039] The term “heteroatoms” shall include oxygen, sulfur and nitrogen.

[0040] The compounds of formula I may form salts with alkali metals suchas sodium, potassium and lithium, with alkaline earth metals such ascalcium and magnesium, with organic bases such as dicyclohexylamine,tributylamine, pyridine and amino acids such as arginine, lysine and thelike. Such salts can be obtained, for example, by exchanging thecarboxylic acid protons, if they contain a carboxylic acid, in compoundsof formula I with the desired ion in a medium in which the saltprecipitates or in an aqueous medium followed by evaporation. Othersalts can be formed as known to those skilled in the art.

[0041] The compounds for formula I form salts with a variety of organicand inorganic acids. Such salts include those formed with hydrogenchloride, hydrogen bromide, methanesulfonic acid, hydroxyethanesulfonicacid, sulfuric acid, acetic acid, trifluoroacetic acid, maleic acid,benzenesulfonic acid, toluenesulfonic acid and various others (e.g.,nitrates, phosphates, borates, tartrates, citrates, succinates,benzoates, ascorbates, salicylates and the like). Such salts are formedby reacting a compound of formula V in an equivalent amount of the acidin a medium in which the salt precipitates or in an aqueous mediumfollowed by evaporation.

[0042] In addition, zwitterions (“inner salts”) are formed.

[0043] Compounds of the formula I may also have prodrug forms. Anycompound that will be converted in vivo to provide the bioactive agent(i.e., the compound for formula I) is a prodrug within the scope andspirit of the invention.

[0044] For example compounds of the formula I may form a carboxylateester moiety. The carboxylate esters are conveniently formed byesterifying any of the carboxylic acid functionalities found on thedisclosed ring structure(s).

[0045] Various forms of prodrugs are well known in the art. For examplesof such prodrug derivatives, see:

[0046] a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985)and Methods in Enzymology, Vol.42, p. 309-396, edited by K. Widder, etal. (Acamedic Press, 1985);

[0047] b) A Textbook of Drug Design and Development, edited byKrosgaard-Larsen and H. Bundgaard, Chapter 5, “Design and Application ofProdrugs,” by H. Bundgaard, p. 113-191 (1991);

[0048] c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);

[0049] d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77,285 (1988); and

[0050] e) N. Kakeya, et al., Chem Phar Bull, 32, 692 (1984).

[0051] It should further be understood that solvates (e.g., hydrates) ofthe compounds of formula I are also within the scope of the presentinvention. Methods of solvation are generally known in the art.

Use and Utility

[0052] The compounds of formula I are microtubule-stabilizing agents.They are thus useful in the treatment of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing;

[0053] carcinoma, including that of the bladder, breast, colon, kidney,liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma;

[0054] hematopoietic tumors of lymphoid lineage, including leukemia,acute lymphocytic leukemia, acute lymphoblastic leukemia, B-celllymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma,hairy cell lymphoma and Burketts lymphoma;

[0055] hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia;

[0056] tumors of mesenchymal origin, including fibrosarcoma andrhabdomyoscarcoma;

[0057] other tumors, including melanoma, seminoma, tetratocarcinoma,neuroblastoma and glioma;

[0058] tumors of the central and peripheral nervous system, includingastrocytoma, neuroblastoma, glioma, and schwannomas;

[0059] tumors of mesenchymal origin, including fibrosarcoma, andrhabdomyoscaroma, and osteosarcoma; and

[0060] other tumors, including melanoma, xenoderma pigmentosum,keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma.

[0061] Compounds of formula I may also inhibit tumor angiogenesis,thereby affecting abnormal cellular proliferation. Suchanti-angiogenesis properties of the compounds of formula I may also beuseful in the treatment of certain forms of blindness related to retinalvascularization, arthritis, especially inflammatory arthritis, multiplesclerosis, restinosis and psoriasis.

[0062] Compounds of formula I may induce or inhibit apoptosis, aphysiological cell death process critical for normal development andhomeostasis. Alterations of apoptotic pathways contribute to thepathogenesis of a variety of human diseases. Compounds of formula I, asmodulators of apoptosis, will be useful in the treatment of a variety ofhuman diseases with aberrations in apoptosis including cancer(particularly, but not limited to follicular lymphomas, carcinomas withp53 mutations, hormone dependent tumors of the breast, prostrate andovary, and precancerous lesions such as familial adenomatous polyposis),viral infections (including but not limited to herpesvirus, poxvirus,Epstein-Barr virus, Sindbis virus and adenovirus), autoimmune diseases(including but not limited to systemic lupus erythematosus, immunemediated glomerulonephritis, rheumatoid arthritis, psoriasis,inflammatory bowel diseases and autoimmune diabetes mellitus),neurodegenerative disorders (including but not limited to Alzheimer'sdisease, AIDS-related dementia, Parkinson's disease, amyotrophic lateralsclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellardegeneration), AIDS, myelodysplastic syndromes, aplastic anemia,ischemic injury associated myocardial infarctions, stroke andreperfusion injury, arrhythmia, atherosclerosis, toxin-induced oralcohol induced liver diseases, hematological diseases (including butnot limited to chronic anemia and aplastic anemia), degenerativediseases of the musculoskeletal system (including but not limited toosteoporosis and arthritis), aspirin-sensitive rhinosinusitis, cysticfibrosis, multiple sclerosis, kidney diseases, and cancer pain.

[0063] The compounds of this invention are also useful in combinationwith known anti-cancer and cytotoxic agents and treatments, includingradiation. If formulated as a fixed dose, such combination productsemploy the compounds of this invention within the dosage range describedbelow and the other pharmaceutically active agent within its approveddosage range. Compounds of formula I can be used sequentially with knownanticancer or cytotoxic agents and treatment, including radiation when acombination formulation is inappropriate. Especially useful arecytotoxic drug combinations wherein the second drug chosen acts in adifferent phase of the cell cycle, e.g. S phase, than the presentcompounds of formula I which exert their effects at the G₂-M phase.

[0064] The present compounds may exist as multiple optical, geometric,and stereoisomers. Included within the present invention are all suchisomers and mixtures thereof

[0065] The compounds of this invention can be formulated with apharmaceutical vehicle or diluent for oral, intravenous or subcutaneousadministration. The pharmaceutical composition can be formulated in aclassical manner using solid or liquid vehicles, diluents and additivesappropriate to the desired mode of administration. Orally, the compoundscan be administered in the form of tablets, capsules, granules, powdersand the like. The compounds are administered in a dosage range of about0.05 to 200 mg/kg/day, preferably less than 100 mg/kg/day, in a singledose or in 2 to 4 divided doses.

Method of Preparation

[0066] A compound of formula I can be prepared as shown in Scheme 1,using procedures described in PCT/EP96/05080. A compound of formula 1.Acan be esterified using, for example, a mixture of formic acid andacetic anhydride to give a corresponding diformate 1.B. A compound offormula 1.C can be prepared from a compound of formula 1.B using a basesuch as DBU. A compound of formula I can be prepared from a compound offormula 1.C by treatment with methanolic ammonia.

[0067] Compounds of formula I where X is O, W is NH, and Q is anoxiranyl group can be prepared as shown in Scheme 2. A compound offormula 2.A can be esterified using, for example, a mixture of formicacid and acetic anhydride to give a corresponding diformate 2.B. Acompound of formula 2.C can be prepared from a compound of formula 2.Bby treatment with a base such as DBU. A compound of formula 2.D can beprepared from a compound of formula 2.C using for examplemethanesulfonyl chloride and triethylamine, or Burgess' reagent.Treatment of a compound of formula 2.D with methanolic ammonia affords acompound of formula I where X is NH and Q is an oxiranyl group.

[0068] A compound of formula 2.A where G is —CR₉{CR₈H can be prepared asshown in Scheme 3. A compound of formula 3.B can be prepared from acompound of formula 3.A (an epothilone or epothilone-related naturalproduct) by formation of pi-allylpalladium complex using, for example,palladium tetrakistriphenylphosphine followed by treatment with sodiumazide (see, for example: Murahashi, S.-I.; et. al., J. Org. Chem. 1989,54, 3292). Subsequent reduction of a compound of formula 3.B with areducing agent such as triphenylphosphine provides a compound of formula3.C. A compound of formula 3.D (or 2.A where G is —CR₉{CR₈H) can beprepared from a compound of formula 3.C by macrolactamization using, forexample, diphenylphosphoryl azide (DPPA) orbromotripyrrolidinophosphonium hexafluorophosphate (PyBroP).

[0069] A compound of formula I where W is O and X is H,H can be preparedas shown in Scheme 4. The alcohol moiety of a compound of formula Iwhere both W and X are O can be protected using methods in the art togive a compound of formula 4.A, where Pl is a suitable O-protectinggroup such as triethylsilyl. Hydrolysis of a compound of formula 4.A,using for example lithium hydroxide monohydrate, provides a compound offormula 4.B. Esterification of a compound of formula 4.B, using forexample trimethylsilyl diazomethane, provides a compound of formula 4.C.Selective dihydroxylation of the α,β-unsaturated ester moiety of acompound of formula 4.C by known methods (see Sharpless, K.B. et al., J.Org. Chem. (1992) 57, 2768) provides a compound of formula 4.D.Oxidative cleavage of the diol of a compound of formula 4.D, using forexample lead tetraacetate provides a compound of formula 4.E. A compoundof formula 4.F can be prepared from a compound of formula 4.E using anallylating agent such as ally bromide and a silver salt such as silveroxide. A compound of formula 4.G can be prepared from a compound offormula 4.F using an olefinating agent such asmethyltriphenylphosphonium bromide and a base such as sodiumhexamethyldisilazide. A compound of formula 4.H can be prepared from acompound of formula 4.G by ring-closing metathesis using either theGrubbs (RuCl₂(=CHPh)(PCY₃)₂; see Grubbs, R. H., et al., Angew. Chem.Int. Ed. Engl.; (1995) 34, 2039) or Schrock catalysts (see Schrock,R.R., et al., J. Am. Chem. Soc., (1990) 112, 3875). A compound offormula I where W is O and X is H,H can be prepared from a compound offormula 4.H by removal of the protecting group using for example aceticacidITHF/water mixtures.

[0070] Alternatively, compounds of formula I where X is H,H can beprepared as shown is Scheme 5. A compound of formula I where X is S canbe prepared from a compound of formula I where X is O using, forexample, Lawesson's reagent[2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide]. Acompound of formula I where X is H,H can be prepared from a compound offormula I where X is S by reduction with reducing agents such astri-n-butyltin hydride, Raney nickel, or nickel boride. In Scheme 5, thehydroxyl group can be optionally protected using, for example, atriethylsily group which can be removed ultimately by treatment withhydrogen fluoride-pyridine or acetic acid/THF/water mixtures.

[0071] Compounds of formula I where Q is an olefinic group or thecorresponding saturated derivative can be prepared as shown in Scheme 6.Compounds of formula I where Q is an oxiranyl group (i.e., compound 6.A)can be reduced using reagents such as reactive titanocene or tungstenchloride and butyllithium to provide compounds of formula I where Q isan olefinic group (i.e., compound 6.B). Further reduction using, forexample, diimide provides compounds of formula I where Q is a saturatedalkyl chain (i.e., compound 6.C).

[0072] In Schemes 1, 3, 4, and 5, the starting material can be obtainedfrom fermentation of SoraiiaLuint cellulosuiii as previously described(see Angew. Chem. Int. Ed. Engl., 1996, 35, No. 13/14.). In thesefermentation products G is usually, but not exclusively, selected fromthe following:

[0073] In cases where G is not selected from the preceding list orobtained from fermentation, synthetic methods can be used. For example,total synthesis routes have been described (See, for example:Danishefsky, S. J., et. al., J. Am. Chem. Soc., (1997) 119, 10073), andthese methods can be used to provide compounds of formula IA where G is,for example, alkyl, substituted alkyl, aryl, substituted aryl,heterocyclo, and —CR₉=CR₈H. In addition, semi-synthesis which utilizesdegradation of natural epothilones can be employed. For example,epothilones (i.e., 7.A) can be protected and then degraded to a compoundof formula 7.B (see PCT/EP96/05080). Subsequent, olefination anddeprotection provides compounds of formula 1.A where is —CCH₃{CR₈H(i.e., compound 7.C). Alternatively, 7.B can be treated with an alkyl orarylmagnesium halide to provide a tertiary alcohol which can bedehydrated using, for example, Burgess reagent to provide a compound offormula 1.A where is —CCH₃{CR₈H (i.e., compound 7.C).

[0074] Furthermore, starting compounds of formula 1.A where G is—C(O)NR₁₀R₁₁ can be prepared from a compound of formula 7.B as shown inScheme 8. A compound of formula 8.A where P is a trialkylsilyl group canbe prepared from a compound of formula 7.B using for examplet-butyldimethylsilyl chloride and triethylamine. Oxidative cleavage of acompound of formula 8.A using for example ozone provides a compound offormula 8.B. Amide coupling of a compound of formula 8.B using methodswell known in the art followed by deprotection provides a compound of1.A where G is —C(O)NR₁₀R₁₁ (i.e., compound 8.C).

[0075] The in vitro assessment of biological activity of the compoundsof formula I was performed as follows:

[0076] In vitro Tubulin Polymerization. Twice cycled (2×) calf braintubulin was prepared following the procedure of Williams and Lee (seeWilliams, R. C., Jr., and Lee, J. C. Preparation of tubulin from brain.Methods in Enzymology 85, Pt. D: 376-385, 1982) and stored in liquidnitrogen before use. Quantification of tubulin polymerization potency isaccomplished following a modified procedure of Swindell, et al., (seeSwindell, C. S., Krauss, N. E., Horwitz, S. B., and Ringel, I.Biologically active taxol analogues with deleted A-ring side chainsubstituents and variable C-2′ configurations. J. Med. Chem. 34:1176-1184, 1991). These modifications, in part, result in the expressionof tubulin polymerization potency as an effective concentration for anygiven compound. For this method, different concentrations of compound inpolymerization buffer (0.1M MES, 1 mM EGTA, 0.5 mM MgCl₂, pH 6.6) areadded to tubulin in polymerization buffer at 37° in microcuvette wellsof a Beckman (Beckman Instruments) Model DU 7400 UV spectrophotometer. Afinal microtubule protein concentration of 1.0 mg/ml and compoundconcentration of generally 2.5, 5.0, and 10 μM are used. Initial slopesof OD change measured every 10 seconds were calculated by the programaccompanying the instrument after initial and final times of the linearregion encompussing at least 3 time points were manually defined. Underthese conditions linear variances were generally<10⁻⁶, slopes rangedfrom 0.03 to 0.002 absorbance unit/minute, and maximum absorbance was0.15 absorbance units. Effective concentration (EC_(0.01)) is defined asthe interpolated concentration capable of inducing an initial slope of0.01 OD/minute rate and is calculated using the formula:EC_(0.01)=concentration/slope. EC_(0.01) values are expressed as themean with standard deviation obtained from 3 different concentrations.EC_(0.01) values for the compounds in this invention fall in the range0.01-1000 μM.

[0077] Cytoxicity (In-Vitro)

[0078] Cytoxicity was assessed in HCT-116 human colon carcinoma cells byMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphenyl)-2H-tetrazolium,inner salt) assay as reported in T. L. Riss, et. al., “Comparison ofMTT, XTT, and a novel tetrazolium compound MTS for in vitroproliferation and chemosensitivity assays.,” Mol. Biol. Cell 3(Suppl.):184a, 1992. Cells were plated at 4,000 cell/well in 96 wellmicrotiter plates and 24 hours later drugs were added and serialdiluted. The cells were incubated at 37° form 72 hours at which time thetetrazolium dye, MTS at 333 μg/ml (final concentration), in combinationwith the electron coupling agent phenazine methosulfate at 25 μM (finalconcentration) was added. A dehydrogenase enzyme in live cells reducesthe MTS to a form that absorbs light at 492 nM which can be quantitatedspectrophotometrically. The greater the absorbance the greater thenumber of live cells. The results are expressed as an IC₅₀, which is thedrug concentration required to inhibit cell proliferation (i.e.absorbance at 450 nM) to 50% of that of untreated control cells. TheIC₅₀ values for compounds of this invention fall in the range 0.01-1000nM.

Preferred Compounds

[0079] As preferred compounds of the present invention are compounds offormula I wherein

[0080] Q is selected from the group consisting of

[0081] and y is oxygen.

EXAMPLE 1

[0082]

[0083][1S-[1R*,3R*(E),7R*,10S*,11R*,12R*,16S*]]-11-Hydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-hydroxymethyl4-thiazolyl)ethenyl]-4,17-dioxabicyclo[14.1.0]heptadec-6(E)-ene-5,9-dione.

[0084] A.[1S-[1R*,3R*(E),7R*,10R*,11S*,12R*,16S*1]]-7,11-Dihydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-methyl4-thiazolyl)ethenyl]-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione, N-oxide.

[0085] A solution of epothilone B (2.0 g, 3.9 mmol) in CH₂Cl₂ (30 mL)was treated with 3-chloroperoxybenwic acid (1.0 g, 5.9 mmol) at 25° C.,under Ar for 2 h. An additional 0.5 g (3.0 mmol) of3-chloroperoxybenzoic acid was added and the reaction mixture was thenstirred for 2 h. The reaction mixture was filtered and the filtrate wasconcentrated in uacuo. The residue was dissolved in EtOAc (100 mL),washed with saturated aqueous NaHCO₃ (75 mL), 5 % aqueous Na₂SO₃ (75mL), H₂O (75 mL), dried (Na₂SO₄) and concentrated in vacuo. The residuewas purified by flash chromatography (SiO₂, 4.5×30 cm, 2-10% MeOH-CHCl₃gradient elution) to afford Compound A (1.04 g, 50%) as a white solid.MS (ESI+): 524.3 (M+H)⁺; MS (ESI−): 522.5 (M−H)⁻.

[0086] B.[1S-[1R*,3R*(E),7R*,10S*,11R*,12R*,16S*]]-7,11-Dihydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-acetoxymethyl4-thiazolyl)ethenyl]-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione.To a resealable Kontes vial was added compound A (0.20 g, 0.38 mmol) andacetic anhydride (2 mL) under Ar. The reaction vessel was sealed underAr and heated to 75° C. for 4 min. Acetic acid (0.4 mL) was thenintroduced into the reaction vessel and the reaction mixture was heatedfor an additional 30 min at 75° C. After the Kontes vial was cooled to25° C., the volatiles were removed it vacuo and the residue was purifiedby flash chromatography (SiO₂, 3.0×15 cm, 45:45:10 hexane/tert-butylmethyl ether/MeOH) to afford Compound B (0.15 g, 68%) as a colorlessoil. MS (ESI+): 566.2 (M+H)⁺, 1131.5 (2M+H)⁺; MS (ESI⁻): 564.4 (M−H)⁻,1129.7 (2M−H)⁻.

[0087] C.[1S-[1R*,3R*(E),7R*,10S*,11R*,12R*,16S*]]-7,11-Dihydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-acetoxymethyl4-thiazolyl)ethenyl]-4,17-dioxabicyclo [14.1.0]heptadecane-5,9-dione, 7,11 diformate.

[0088] A solution of compound B (0.15 g, 0.27 mmol) in CH₂Cl₂ (5 mL) wastreated with 4-N,N-dimethylaminopyridine (71 mg, 58 mmol), triethylamine(0.37 mL, 2.6 mmol), and formic acid (50 mL, 1.3 mmol) at 25° C., underAr. The reaction mixture was cooled to −15° C. and acetic anhydride(0.12 mL, 1.3 mmol) was added over 3 min. The reaction mixture wasstirred at −15° C. (15 min), warmed to 25° C. (15 min), quenched with pH7.0 phosphate buffer and extracted with EtOAc (3×30 mL). The combinedorganic extracts were washed with aqueous 1 N HCl (50 mL), 10% aqueousNaHCO₃ solution (50 mL), brine (50 mL), dried (Na₂SO₄), and concentratedin vacuo. The residue was purified by flash chromatography (SiO₂, 1.5×10cm, 10% acetone-CH₂Cl₂) to afford Compound C (0.134 g, 84%) as a glass.MS (ESI⁺): 622.2 (M+H)⁺.

[0089] D.[1S-[1R*,3R*(E),7R*,10S*,11R*,12R*,16S*]]-11-Hydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-acetoxymethyl4-thiazolyl)ethenyl]4,17-dioxabicyclo[14.1.0] heptadee-6(E)-ene-5,9-dione, 11-formate.

[0090] A solution of compound C (0.13 g, 0.21 mmol) in CH₂Cl₂ (2.2 mL)was treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (0.31 mL, 2.1 mmol)at 25° C., under Ar. The reaction mixture was stirred at 25° C., for 2h, quenched by the addition of pH 4.0 phosphate buffer, and extractedwith EtOAc (3×20 mL). The combined organic extracts were washed withsaturated aqueous NaHCO₃ solution (30 mL), brine (30 mL), dried(Na₂SO₄), and concentrated in vacuo. The residue was purified by flashchromatography (SiO₂, 1.5×10 cm, 25-50% EtOAc-hexane gradient elution)to afford Compound D (0.11 g, 92%) as a foam. MS (ESI⁺): 576.2 (M+H)⁺.

[0091] E.[1S-[1R*,3R*(E),7R*,10S*,11R*,12R*,16S*]]-11-Hydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-hydroxymethyl-4-thiazolyl)ethenyl]4,17-dioxabicyclo[14.1.0]heptadec-6(E)ene-5,9-dione.

[0092] A solution of compound D (0.11 g, 0.19 mmol) in MeOH (1.0 mL) wastreated with 2 M ammonia in methanol (1.0 mL) at 25° C., under Ar. Thereaction mixture was warmed to 45° C. for 1 h and concentrated in vacuo.The residue was purified by flash chromatography (SiO₂, 1.5×10 cm, 2-5%MeOH-CHCl₃ gradient elution) to afford the title compound (95 mg, 98%)as a white foam. MS (ESI⁺): 506.2 (M+H)⁺, 1011.3 (2M+H)⁺; MS (ESI⁻):504.5 (M−H)⁻.

EXAMPLE 2

[0093]

[0094][1S-[1R*,3R*(E),7R*,10S*,11R*,12R*,16S*]]-11-Hydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-azidomethyl-4-thiazolyl)ethenyl]-4,17-dioxabicyclo[14.1.0]heptadec-6(E)-ene-5,9-dione.

[0095] To a stirred solution of Compound 1E (3.0 mg, 0.0059 mmol) in 0.5mL THF at 0° C. was added a 0.2M solution of diphenylphosphoryl azide(DPPA) in THF (35 μl, 0.0071 mmol, 1.2 eq) followed by addition of a0.2M solution of DBU in THF (30 μL, 0.0060 mmol, 1 eq). The mixture wasallowed to stir at OC for 3.5 h. An additional 15 μL of DPPA solution(0.0030 mmol, 0.5 eq) and 30 μL of DBU solution (0.0060 mmole, 1 eq)were added, and the mixture was allowed to stir at 0° C. for anadditional 20 min. The solution was then warmed to 25C and allowed tostir for 15 h. The mixture was diluted with 60 mL ethyl acetate thenwashed with 10 mL water and dried over Na₂SO₄. The organic layer wasconcentrated in vacuo and purified by silica gel chromatography using2.5% MeOH in CHCl3 to afford 2 mg of a clear film (65%). M+H=531.2

[0096] add name to claims:

[0097][1S-[1R*,3R*(E),7R*,10S*,11R*,12R*,16S*]]-11-Hydroxy-8,8,10,12,16-pentamethyl-3-[1-methyl-2-(2-arninomethyl-4-thiazolyl)ethenyl]-4,17-dioxabicyclo[14.1.0]heptadec-6(E)-ene-5,9-dione.

What is claimed:
 1. A compound of the formula

wherein Q is selected from the group consisting of

G is selected from G is selected from the group consisting of alkyl,substituted alkyl, aryl, substituted aryl, heterocyclo,

W is O or N R₁₂; Xis O, S, or H, H; Y is selected from the groupconsisting of O; H, OR₁₃; OR₁₄, OR₁₄; NOR₁₅; H, NOR₁₆; H, NR₁₇R₁₈; H, H;or CHR₁₉; OR_(14,) OR₁₄ can be a cyclic ketal; B is selected from thegroup consisting of H, OR₂₀, or OCOR₂₁, and NR₂₂R₂₃; D is selected fromthe group consisting of NR₂₄R₂₅ and saturated heterocycle (such aspiperidinyl, pyrrolidinyl, and the like); R₁, R₂, R₃, and R₄ areselected from H or lower alkyl; R₁₅, R₁₆, R₁₇, R₁₈, and R₁₉ are selectedfrom the group H, alkyl, substituted alkyl and aryl; R₆, R₇, R₁₃, R₁₄,R₂₀ and R₂₁ are selected from the group H, alkyl and substituted alkyl;R₅, R₈, R₉, R₂₂, R₂₄, R₂₆ and R₂₇ are selected from the group consistingof H, alkyl, substituted alkyl, aryl, heteroaryl, cycloalkyl andheterocyclo; R₁₂, R₂₃ and R₂₅ are selected from the group consisting ofH, alkyl, substituted alkyl, aryl, heteroaryl, cycloalkyl, heterocyclo,R₂₆C{O, R₂₇SO₂, hydroxy, O-alkyl or O-substituted alkyl; and any salts,solvates or hydrates thereof with the proviso that together W and X asO; R₁, R₂, R₃, R₄ as methyl; R₅ as H or methyl; G as1-methyl-2-([2-]methyl-4-thiazolyl)ethenyl; and Q as

are excluded.
 2. The compound of claim 1 wherein Q is selected from thegroup consisting of

and y is oxygen.
 3. A compound of the formula